p16INK4a in Cochlear Hair Cell Regeneration.

描述(申请人提供)：听力损失主要是由内耳耳蜗区的感觉毛细胞(HC)受损引起的。人类和其他哺乳动物不能替代受损的HCS；然而，鸡、鱼和两栖动物可以通过邻近支持细胞(SC)的增殖和转分化来替代(Conwin和Oberholtzer，1997)。有趣的是，肿瘤抑制基因p16INK4a在具有再生HCs能力的非哺乳动物脊椎动物中缺失(Kim等人，2003；Gil和Peters，2006)。在哺乳动物中，p16INK4a作为一种细胞周期蛋白依赖性的激酶抑制因子，使细胞处于静止状态。它的表达是由AGE和有丝分裂信号诱导的(Zindy等人，1997)。众所周知，p16INK4a在包括神经元在内的成年细胞的体内再生能力中也发挥着关键作用(Janzen等人，2006年；Molofsky等人，2006年)。通常认为，破坏了HCS释放的信号，导致SCs重新进入细胞周期。在非哺乳动物脊椎动物中，这会导致HC再生；然而，在哺乳动物中，我们预测这种有丝分裂活动诱导p16INK4a的表达，使SCs保持静止状态，从而阻止HC再生。有没有可能赋予小鼠，鸡通过灭活p16INK4a来再生HCS的能力？为了实现这一目标，我们提出了以下中心假设：在哺乳动物中，p16INK4a的失活将允许干细胞：(1)对抗生素或基因消融损伤的HCs释放的信号做出反应，(2)重新进入细胞周期，(3)再生HCs。为了验证这一假设，我们提出了以下具体目标：具体目标1：确定p161NK4a基因缺失小鼠在氨基糖苷类抗生素庆大霉素引起的HC损伤后干细胞的再生能力。特定目的2：检测p16INK4a基因缺失小鼠在出生后HCS中视网膜母细胞瘤蛋白急性失活引起的HC损伤后，干细胞的再生能力。虽然哺乳动物不能自发地再生HC，但通过对耳蜗SCs的遗传和/或治疗操作，可以诱导类似于非哺乳动物脊椎动物的HC再生过程。这项提案是朝着恢复那些接触耳毒性药物、噪音或其他环境物质的人的听力的最终目标迈出的第一步。此外，出生后HCS中视网膜母细胞瘤蛋白的急性失活导致的HC损伤是该领域的一项技术突破。

DESCRIPTION (provided by applicant): Hearing loss is primarily caused by damage to sensory hair cells (HC) in the cochlea of the inner ear. Humans and other mammals cannot replace damaged HCs; however, chicken, fish and amphibians can, by proliferation and transdifferentiation of neighboring supporting cells (SC) (Conwin and Oberholtzer, 1997). Interestingly, the tumor suppressor gene, p16INK4a, is absent in non-mammalian vertebrates that have the capacity to regenerate HCs (Kim et al., 2003; Gil and Peters, 2006). In mammals, p16INK4a acts as a cyclin-dependent kinase inhibitor that keeps cells in a quiescent state. Its expression is induced by age as well as mitogenic signaling (Zindy et al., 1997). p16INK4a is also known to play a critical role in the in vivo regenerative ability of adult cells, including neurons (Janzen et al., 2006; Molofsky et al., 2006). It is commonly thought that damaged HCs release signals that cause SCs to reenter the cell cycle. In non-mammalian vertebrates this results in HC regeneration; however, in mammals, we predict that this mitotic activity induces the expression of p16INK4a which keeps SCs in a quiescent state and thus prevents HC regeneration. Is it possible to give a mouse, the chicken's ability to regenerate HCs by inactivating p16INK4a? To achieve this goal, we propose the following central hypothesis: Inactivation of p16INK4a in mammals will allow SCs: (1) to respond to signals released from HCs damaged by antibiotics or genetic ablation, (2) to reenter the cell cycle and (3) to regenerate HCs. To test this hypothesis, we propose the following specific aims: Specific Aim 1: To determine the regenerative capacity of SCs in p161NK4a-null mice after HC damage caused by the aminoglycoside antibiotic, gentamicin. Specific Aim 2: To determine the regenerative capacity of SCs in p16INK4a-null mice after HC damage caused by acute inactivation of the retinoblastoma protein in postnatal HCs. Although mammals cannot spontaneously regenerate HCs, a process of HC regeneration similar to what occurs in non-mammalian vertebrates could be induced by genetic and/or therapeutic manipulations of cochlear SCs. This proposal is the first step toward the final goal of restoring hearing in those exposed to ototoxic drugs, loud noise or other environmental agents. In addition, HC damage induced by acute inactivation of the retinoblastoma protein in postnatal HCs is a technological breakthrough for the field.